Lipocortin-I is a known protein, of relative molecular weight of about 35 kilodaltons and conserved amino acid sequence, found in avian and mammalian species. The protein has been referred to by several names, in addition to lipocortin-I, including "p35," on account of its molecular weight, and "calpactin-II, " due to its ability to bind calcium, phospholipid and acting, in a manner similar to calpactin-I, discussed below. In the present specification, the protein will be referred to as "lipocortin-I. " The amino acid sequence of human lipocortin-I is provided in Wallner et al., Nature, 320, 77-81 (1986), and in Patent Cooperation Treaty Application Publication No. 86-04094, published July 17, 1986.
Lipocortin-I is a substrate for phosphorylation by protein kinase C and the protein-tyrosine kinase, epidermal growth factor receptor. Lipocortin-I is found in the same cell types as calpactin-I but, unlike calpactin-I-heavy chain, is potentially secreted from such cells, including macrophages in response to corticosteroids.
Lipocortin-I inhibits the enzyme phospholipase A.sub.2. See Wallner et al., supra, and PCT Application Publication No. 86-04094.
Calpactin-I-heavy chain is a known protein, of relative molecular weight of about 36 kilodaltons and highly conserved amino acid sequence found in high concentration intracellularly in certain cells, especially epithelial and endothelial cells, of avian and mammalian species. The amino acid sequence of calpactin-I-heavy chain is highly homologous with that of lipocortin-I, and the two proteins have similar properties and functions.
Calpactin-I-heavy chain has been referred to by various names, including "p36," on account of its molecular weight, and "lipocortin-II" on account of the numerous properties it has in common with lipocortin-I, discussed above. In the present specification, calpactin-I-heavy chain will be referred to as "lipocortin-II." A partial amino acid sequence of human lipocortin-II is provided in PCT Application Publication No. 86-04094, wherein the protein is referred to as "N-lipocortin," and in Huang et al., Cell, 46, 191-199 (1986). The sequence of lipocortin-II (p36) is also provided in Saris et al., Cell, 46, 201-212 (1986), and reveals a multidomain protein with internal repeats.
Lipocortin-II is a substrate for phosphorylation by protein kinase C, retroviral protein-tyrosine kinases and other protein-tyrosine kinases, including epidermal growth factor receptor and platelet-derived growth factor receptor.
The protein, in association with the 11-kilodalton calpactin-I-light chain, forms calpactin-I, which binds calcium, phospholipid, actin and spectrin. Calpactin-I is associated with the cellular plasma membrane as well as the cortical skeleton underlying the plasma membrane and is thought to play a structural role in the cortical skeleton.
Like lipocortin-I, lipocortin-II can act as an inhibitor of phospholipase A.sub.2. See Huang et al., supra, and PCT Application Publication No. 86-04094.
Given the similarity of properties and functions of lipocortin-I and lipocortin-II, the isolation of antibodies which are specific to one but not both of the proteins has been a problem which, until the present invention, was not solved. Indeed, at the time of the present invention, it was not apparent that such antibodies could be isolated.
Phospholipase A.sub.2 is a key enzyme in providing arachidonic acid for the arachidonic acid cascade, wherein prostaglandins, thromboxanes, and leukotrienes are produced. These compounds have a number of physiological effects, including inflammation.
A number of anti-inflammatory therapeutic agents, such as corticosteroids, are known Which act by directly or indirectly, through intermediates induced by the agents, inhibiting phospholipase A.sub.2, and, consequently, the production of arachidonic acid and other compounds of the arachidonic acid cascade. Lipocortin-I and lipocortin-II are similarly useful therapeutically as anti-inflammatory agents by virtue of their ability to directly inhibit phospholipase A.sub.2. The anti-inflammatory activity of lipocortin-I and lipocortin-II might also be mediated by their blocking the chemotactic movement of neutrophils and macrophages into inflamed tissues. See, e.g., PCT Application Publication No. 86-04094.
Levels of lipocortin-I and lipocortin-II are important in control of inflammation. Thus, the ability to assay for levels of lipocortin-I and lipocortin-II in tissues and body fluids, such as lymph and serum, of a person suffering from a disorder involving inflammation, such as various arthritic, allergic, dermatologic, ophthalmic, and collagen diseases, would provide a useful diagnostic tool for understanding the cause of the patient's disease and selecting therapy appropriate to treat the disease effectively.